September 2013

September 30, 2013

In today’s
new issue of JCB, Lerit and Rusan
describe how and why the centrosomes of Drosophila
neuroblasts show asymmetric levels of microtubule nucleation and organizing
activity. As described in this week’s In Focus, the pericentrin orthologue PLP
is enriched on the older, mother centrosome during interphase, and limits its
activity by restricting the recruitment of Polo kinase, the master regulator of
centrosome maturation. In cells lacking PLP, both centrosomes are equally
active, which inhibits their ability to separate to opposite sides of the cell
and segregate into different daughter cells during mitosis.

Meanwhile,
Horn et al. identify a protein that helps homologous chromosomes pair up in
meiosis by connecting them to the microtubule cytoskeleton. The outer nuclear
membrane protein KASH5 links the cytoplasmic motor protein dynein to the inner
nuclear membrane protein SUN1, which, in turn, connects to the telomeres of
meiotic chromosomes. Dynein can therefore pull the chromosomes into clusters to
facilitate homolog pairing and recombination. But this process fails in mice
lacking KASH5, leading to meiotic arrest and infertility. More here.

And Luo
et al. describe the organization and dynamics of an actomyosin network that may
help maintain cell shape. As author Alexander Bershadsky explains in this
month’sbiosights video, the network is formed by actin nodes that contain the
formin DAAM1 and the crosslinker filamin A, and are connected to each other by
myosin II. You can watch the video below, or in iTunes, and, if you’d like to
use it as the basis for your next journal club, you can download the video as
part of our Journal Club pack.

September 28, 2013

The Keynote lecture on Day 3 was from Hans Clevers,
who is director of, and runs a lab at, the Hubrecht Institute in Utrecht and is
also President of the Royal Netherlands Academy of Arts and Sciences. Clevers gave an amazing presentation of his
lab’s work on intestinal stem cells that was accompanied by movie-quality
animations that really helped visualize the significance of his results. The intestinal epithelium has very
interesting morphology: we all know about villi and their importance in
digestion but in between the villi are ‘crypts’, narrow tunnels at the bottom
of which lie the intestine’s stem cells.
Each crypt makes 200 cells/day so this is a very active stem cell
compartment. It has to be because the cells of this epithelium must be replaced
every 4-5 days. Defining the stem cell
population in the crypt has been challenging and the subject of much
controversy. Clevers’ lab has made great
strides in this area using sophisticated genetic engineering techniques that
allow them to mark and manipulate single cells in the mouse intestine (for a great review
on lineage tracing in epithelia, click here).
They identified a population of columnar epithelial cells at the bottom
of the crypt, positive for the marker Lgr5 (see figure), that give rise to all the cell
types of the intestine.
The surrounding Paneth cells serve as the niche
for these stem cells.
They isolated these Lgr5-positive cells, figured out the conditions to grow
them in vitro and found, amazingly, that these single cells could grow into
‘miniguts’ with the right tissue morphology. They then introduced these miniguts into mice
and found that they attached only to damaged epithelia (which occurs for
example with ulcers) and sealed the lesion, without any occurrence of adenomas. These miniguts have great therapeutic
potential; Clevers showed that they can
grow miniguts from intestinal stem cells isolated through patient biopsies and
test the efficacy of drugs on this pseudotissue. For example, they have used the miniguts to
monitor CFTR channel function and test cystic fibrosis drugs. I found this to be a very inspiring
talk: this lab came to this field
through a long standing interest in Wnt signaling and now are actively
exploring the therapeutic potential of their findings. It really was a tour-de-force example of how
basic cell biology can lead to more clinical applications.

September 24, 2013

Carolyn Moores kicked things off by describing how her lab
uses cryo-electron microscopy to determine the structures of microtubules and
their associated regulatory factors. You might wonder how examining the static
structures of microtubules tells you anything about their dynamics. But
identifying how microtubule-associated proteins bind to microtubules reveals a
great deal about their function. In a 2010 JCB
paper, for example, Moores and colleagues showed that the
microtubule-stabilizing protein doublecortin binds at the intersection of four
tubulin heterodimers, revealing how the protein can stabilize both lateral and
longitudinal contacts between the microtubule subunits. And, in 2012,
Moores and colleagues showed how microtubule plus-end tracking proteins (+TIPs)
of the EB family bind close to the tubulin GTPase site, suggesting how the
proteins might be able to sense the nucleotide-binding state of tubulin
subunits at the growing ends of microtubules.

Many additional +TIPs bind to EB proteins at the plus ends
of microtubules through a core motif containing the amino acid sequence SxIP. Anna Akhmanova described her lab’s
recent proteomic screen to identify new +TIPs that contain this motif.
According to Akhmanova, almost every cellular structure that interacts with
microtubules has its own +TIP.

The session’s chairperson, Marileen Dogterom, discussed her
lab’s approach to studying cytoskeletal dynamics, in which they attempt to
reconstitute microtubule behavior in minimal, in vitro systems. They’ve
successfully used this approach to study how cortical dynein positions
microtubule asters, and her group is currently looking at how microtubules
cooperate with the actin cytoskeleton.

September 23, 2013

The talks covered transport (and interactions) between many
different membrane compartments. Ludger Johannes, the session chair, discussed
the mechanism of clathrin-independent endocytosis, describing how the plasma
membrane can bud inwards in the absence of coat proteins like clathrin or
caveolin. Jacques Neefjes, on the other hand, discussed how vesicles containing
MHC Class II molecules are transported to the surface of antigen-presenting
cells.

Diana Zala (from Frederic Saudou's lab) described her recent paper about the rapid
transport of vesicles along motor axons. The vesicles are moved by
microtubule-based motor proteins and, given the enormous length of motor axons,
this requires an awful lot of energy. Zala and colleagues found that this
energy is provided by the glycolysis reaction, which can take place on the
surface of the vesicles themselves due to the recruitment of glycolytic enzymes
like GAPDH. In the absence of vesicular GAPDH, the vesicles are transported
along axons much more slowly, indicating that rapid and efficient axonal
transport requires the vesicles to be “energy-independent”.

Antonella De Matteis discussed the non-vesicular transport
of the lipid glucosylceramide, a key precursor of glycosphingolipids.
Glucosylceramide is transported through the Golgi by the lipid transfer protein
FAPP2, which extracts glucosylceramide from the cis-Golgi membrane and deposits
it at the trans-Golgi network (TGN). De Matteis showed how binding to
glucosylceramide increases FAPP2’s affinity for the phospholipid PI(4)P, which
helps target the protein to the TGN. Intriguingly, glucosylceramide can also be
transported through the Golgi stack via a vesicle-based mechanism. De Matteis
showed FAPP2 is converted into different types of glycosphingolipid, depending
on whether it passes through each Golgi cisterna in turn, or whether it is
transported directly to the TGN by FAPP2.

And finally, Francesca Giordano (from Pietro De Camilli's laboratory) described how a family of
proteins called extended synaptotagmins helps connect the endoplasmic reticulum
to the plasma membrane. These proteins localize to contact sites and contacts are
lost when the extended synaptotagmins are depleted from human cells. However, calcium
homeostasis (which is thought to be the main function of ER-plasma membrane
contacts) isn’t affected in the absence of the extended synaptotagmins, so the physiological
function of these proteins remains unclear.

September 22, 2013

The 2013 EMBO meeting in Amsterdam opened on Saturday night
with a thought-provoking plenary session on cellular organization. Several of
the talks focused on the emerging idea that both cellular membranes and the
cytoplasm can be compartmentalized by the physical process of phase separation.

One of the original examples of this phenomenon is, of
course, the idea of lipid rafts in the plasma membrane. Rafts form when sphingolipids
and cholesterol (which form a liquid-ordered bilayer) separate out from
phospholipids in the liquid-disordered phase. By recruiting specific membrane
proteins, this compartmentalization of the membrane can promote numerous
processes such as cell signaling pathways.

The pioneer of this idea, Kai Simons, gave the opening
lecture of the session and discussed his recent efforts (including this 2012 JCB paper) to prove the existence of
lipid rafts in vivo, a difficult proposition give the tiny size and transient
nature of rafts. He also described his recent finding that bacteria – which
lack sterols – may instead use a class of molecules called hopanoids to form
regions of liquid-ordered membrane and phase separation. In Simons’ view, the
plasma membrane is “poised for phase separation” and compartmentalization, but
emerging data suggests that the cytoplasm may be similarly poised.

Tony Hyman (one of the conference chairs) pointed out in his
talk that many cytoplasmic reactions are compartmentalized without the aid of
membranes. (For example, the nucleolus contains over 100 proteins involved in
making ribosomes). Hyman began is presentation by describing the behavior of
cytoplasmic RNA and protein particles called P granules. In 2009, Hyman and
colleagues showed that, in C. elegans
embryos, P granules behave like liquid droplets within the cytoplasm. (Picture a separated vinaigrette in which drops of vinegar have separated from the oil). Specifically, P granules seem to be colloidal liquids formed by multivalent, weak
interactions between the P granule components. P granules preferentially form
at the posterior of C. elegans
embryos because the transition to the liquid phase is favored at this end of
the embryo. And, although the P granule components can freely diffuse within
the granule itself, their diffusion across the phase boundary into the aqueous
solution phase of the cytoplasm is limited. This helps the P
granule components stay together. More recently, Hyman and colleagues have shown
that nucleoli behave like liquids as well.

In the following talk, Michael Rosen extended the idea of
phase separation to two dimensional structures that form on membranes, such as
the network of nephrin, NCK, and N-WASP that induces actin polymerization at
the plasma membrane. Again, these components form a large number of multivalent
interactions (boosted by phosphorylation of the nephrin receptor), resulting in
a transition to the liquid phase and separation from the surrounding aqueous
phase. This separation helps activate the Arp2/3 complex to initiate
actin polymerization.

Rosen and Hyman think that similar principles could apply to
many cellular structures. It’s certainly an interesting and important way to
think about cellular function but, as the speakers themselves pointed out, it
isn’t necessarily a new way to look at things. In the first half of the
twentieth century – before molecular biology allowed researchers to identify
and manipulate individual genes and proteins – scientists were more concerned
with the physicochemical processes that drive cell function. It seems that one
of those processes, particularly with respect to cellular organization, may be
phase transitions and phase separation.

September 16, 2013

In today’s
new issue of JCB, Niessen et al.
describe how atypical protein kinase C regulates the differentiation of
epidermal stem cells by controlling the balance between symmetric and
asymmetric cell divisions. As described in this week’s In Focus, mice lacking
atypical protein kinase C from their epidermis suffer premature aging and hair
loss.

Xu
et al. reveal that the phospholipid translocase Drs2 flips phosphatidylserine
molecules from the inner to the outer leaflet of cellular membranes in order to
promote vesicle transport between the trans-Golgi network and early endosomes.
Insertion of this phospholipid into the outer leaflet increases the negative
charge and curvature of membranes, thereby recruiting a protein called Gcs1 to
the trans-Golgi network and early endosomes to stimulate transport between the
two organelles. More here.

Nishimura
et al. generate silicon nanocrystals that can be used as probes for single
molecule fluorescence microscopy. As explained here, the crystals are small and
emit red light without photobleaching or blinking, allowing the researchers to
track the endocytosis of individual transferrin receptors.

Meanwhile,
Ando et al. describe how the small GTPase Rap1 strengthens endothelial
cell-cell junctions by controlling the activation of myosin II (see this
summary for more), and Madsen et al. reveal how a population of M2-like
macrophages supports tissue remodeling by taking up and degrading the
extracellular matrix protein collagen. You can learn more about this latter
story by listening to this month’s biobytes podcast, where you’ll also hear
Dileep Varma describe his recent study mapping the precise location of spindle checkpoint
proteins within kinetochores. You can listen below or subscribe in iTunes.

That’s
all for today but, as always, there are plenty more interesting stories for
you to discover on our table of contents, which you can visit by clicking here.

September 02, 2013

In the
latest issue of JCB, Hung et al. reveal
how cells use different mechanisms to migrate through large or small spaces. The
authors demonstrate that, in spacious surroundings, cell movement depends on
the small GTPase Rac1, which inhibits myosin II and stimulates membrane
protrusion. In confined spaces, however, Rac1 is inactive and cell migration is
driven by myosin II activity. You can learn how the focal adhesion protein paxillin
controls this switch in this week’s In Focus.

And Worth
et al. reveal how the cyclin-dependent kinase Cdk5 activates a protein called
drebrin, which organizes actin filaments and links them to microtubules in
neuronal growth cones. As explained here, the researchers show that Cdk5
phosphorylation triggers a conformational change in drebrin that uncovers the
protein’s actin-bundling domains.

Meanwhile,
Yi et al. describe how T cells move their centrosomes to the immunological
synapse that connects them to antigen-presenting cells via a biphasic process
involving the dynein-dependent capture and depolymerization of microtubules.
Senior author John Hammer discusses his lab’s work in this month’s biosights
video podcast. (You can watch below or subscribe in iTunes). And if you think
you might like to present this paper in your next journal club, don’t forget
you can download everything you need in our Journal Club Pack here.

And finally, a quick note from the JCB's reviews editor, Priya Prakash Budde, about our latest review series, which kicks off in today's new issue...

In this issue,
we debut the first review of the series that we will package into a special
issue for distribution at the annual ASCB meeting. This year, the series is on 'cell
biology in neuroscience', which covers the cell biology underlying various
topics in brain development, function and homeostasis. The first review from
Jonathan Cooper is on cell migration in the developing nervous system.
One of the great feats in brain development is the long range migration
that neurons undergo to get to their final destination. Cooper does a
wonderful job of describing the cellular basis of how neurons perform this
migration. Two key issues described in the review are that there are some
unique aspects to the machinery used by neurons to migrate, and that neurons
display a remarkable diversity in their modes of migration. It is a
really well-written, accessible review that brings out both unifying themes and
important distinctions in how cell migration occurs in these cells.

In
the issues leading up to the ASCB meeting, we will publish the other reviews in
the series that will cover the cellular basis of axon growth, guidance, synapse
formation, synaptic plasticity, cell death in the brain and on the role of glia
in neuronal development. In covering this range of topics, what struck me
is that the reviews cover quintessential cell biological phenomena, such as
cell migration, cell polarity, cytoskeletal dynamics, signaling in subcellular compartments,
interactions between cells and the extracellular environment, cell death and
cell-cell communication. So there is something for every cell biologist
in the series and we hope you enjoy it.

Last, I want to thank former JCB
editorial board member Josh Sanes, and JCB's Senior Editor Louis Reichardt for
their invaluable help in crystallizing the topics and authors for this series.

That's all for today, but you can find plenty more interesting papers listed on our
table of contents page here.

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